Abstract:
Objective To clone human tumstatin gene, express its recombinant protein for further research. Methods Total RNA was extracted from HEK293 cell. The tumstatin gene was amplified by RT-PCR, then cloned into pMD19-T and sequenced. Subsequently, the tumstatin gene was cloned into the p ET28a and t ransformed into E. coli BL21 (DE3) where it was induced to express by IPTG. The products were identified by SDS-PA GE and Western blot . Results RT-PCR product was about 750bp, its sequence was the same as that of tumstatin reported. The expression vector pET28a-tumstatin was constructed successfully, and there was a new protein band about Mr 29 KD on SDS-PAGE. The ratio of the expressed product to total bacterial proteins was 30 %. The result of Western blot demonst rated that the expressed product was the tumstatin protein. Conclusion Human tumstatin gene was cloned and its recombinant proteins were expressed successfully in this study.
下载:
